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Sample GSM4886997 Query DataSets for GSM4886997
Status Public on Jan 07, 2022
Title sgRNAseq of CRISPR TF transduced ESCs on day 8 post-transduction
Sample type SRA
 
Source name Embryonic stem cells
Organism Mus musculus
Characteristics genotype: Ainv15(iNgn2 Tubb3::GFP)
treatment: No treatment
cell type: Embryonic stem cells
Treatment protocol 50 ng/µL Zeocin (Invivogen) was added to each well 24h later and transduced cells were maintained with Zeocin selection. To maintain 50-70% confluency throughout the screen, cells were transferred to 6-well plates (Thermo Fisher) on day 2 and selected for 3 additional days for a total of 5 days of selection. The first time point was obtained on day 5 post-selection. The second and third timepoints were taken on day 8 and day 12 while growing in ESC medium containing 50 ng/µL Zeocin.
Growth protocol 3 x 10^6 ESC cells were distributed to 5 12-well dishes (Thermo Scientific) with 5 x 10^5 mESC cells per well. 4 h after cells were plated, the media was replaced with 8 µL of 100x concentrated library lentivirus mixed in 1 mL ESC medium with 1x polybrene (EMD Millipore). 16 h later, the virus solution was removed, cells were washed 1 time with PBS1X, and cells were split 1:2 to 10 12-well plates.
Extracted molecule genomic DNA
Extraction protocol In a 15 ml conical tube, we added 6 ml of NK Lysis Buffer (50 mM Tris (Boston BioProducts), 50 mM EDTA (Ambion), 1% SDS (Invitrogen), pH 8), and 30 μl of 20 mg/ml Proteinase K (Qiagen) to 9 million cells (>500 coverage) and incubated at 55°C overnight. 30 μl of 10 mg/ml RNAse A (Qiagen, diluted in NK Lysis Buffer to 10 mg/ml and then stored at 4 °C) was added to the lysed sample, which was then inverted 25 times and incubated at 37°C for 30 minutes. Samples were cooled on ice before the addition of 2 ml of pre-chilled 7.5M ammonium acetate (Sigma) to precipitate proteins. After adding ammonium acetate, the samples were vortexed at high speed for 20 seconds and then centrifuged at ≥ 4,000 x g for 10 minutes. After the spin, a tight pellet was visible in each tube and the supernatant was carefully decanted into a new 15 ml conical tube. 6 ml 100% isopropanol was added to the tube, inverted 50 times and centrifuged at ≥4,000 x g for 10 minutes. Genomic DNA was visible as a small white pellet in each tube. The supernatant was discarded, 6 ml of freshly prepared ice-cold 70% ethanol was added, the tube was inverted 10 times, and then centrifuged at ≥4,000 x g for 1 minute. The supernatant was discarded by pouring; the tube was briefly spun, and the remaining ethanol was removed using a P200 pipette. After air-drying for 10-30 minutes, the DNA changed appearance from a milky white pellet to slightly translucent. At this stage, 500 μl of 1x TE buffer (Sigma) was added, the tube was incubated at 65°C for 1 hour and at room temperature overnight to fully resuspend the DNA. The next day, the gDNA samples were vortexed briefly. The gDNA concentration was measured using a Nanodrop (Thermo Scientific).
5 PCR1 reactions were used for 52 µg of DNA for >500x coverage. Per PCR tube, 10.4 µg of genomic DNA was used in 100 µL volume with 0.4 µL Taq-B Polymerase (Enzymatics), 10X Taq buffer (Enzymatics), 10 mM dNTPs (NEB), 10 µM Fwd (5’ GAGGGCCTATTTCCCATGATTC) and Rvs (5’ GTTGCGAAAAAGAACGTTCACGG) using the following PCR protocol: Denaturation 94 °C for 30s, 25 cycles of 94 °C for 10s, 55 °C for 30s, 68 °C for 45s, final elongation 68 °C for 2 min. For the addition of Illumina barcodes, 5 staggered forward primers and 1 reverse primer with specific barcodes containing Illumina adaptors were mixed to obtain 10 µM primer mix. 2 PCR2 reactions were used per timepoint with 5 µL of PCR1 product amplified with 25 µL Q5 High Fidelity 2X Master Mix (NEB), 5 µL of 10 µM primer mix in 50 µL volume using the following PCR protocol: Denaturation 98 °C for 30s, 10 cycles of 98 °C for 10s, 65 °C for 30s, 72 °C for 45s, final elongation 72 °C for 5 min. All samples were pooled in equimolar ratio and PCR purified pooled sample with QIAquick PCR Purification Kit (Qiagen). The purified sample was then run on a 2% E-Gel and the correct size band was extracted for PCR2 (250-270 bp) and purified with QIAquick Gel Extraction Kit (Qiagen). The pooled library was then quantified with a Low-Range Quantitative ladder on 2% E-Gel. Libraries were then sequenced on Illumina MiSeq v3 for 150 cycles single end at the genomics core facility at NYU.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina MiSeq
 
Description genome-integrated sgRNAs
Day8_CRISPR_TF_transduced_cells
Data processing Library strategy: sgRNA-seq
Sequences flanking the 20 nucleotide sgRNA sequence were trimmed using cutadapt.
Remaining 20 nucleotide sgRNA sequence reads were aligned to a library index generated by 17820 sgRNA sequences using bowtie.
Genome_build: mm10
Supplementary_files_format_and_content: TXT: tab-delimited text file containing output of featurecounts for Refseq genes.
 
Submission date Nov 06, 2020
Last update date Jan 08, 2022
Contact name Shaun Mahony
E-mail(s) mahony@psu.edu
Phone 814-865-3008
Organization name Penn State University
Department Biochemistry & Molecular Biology
Lab Shaun Mahony
Street address 404 South Frear Bldg
City University Park
State/province PA
ZIP/Postal code 16802
Country USA
 
Platform ID GPL16417
Series (2)
GSE160963 The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II at pro-differentiation genes [sgRNA-seq]
GSE160966 The BTB transcription factors ZBTB11 and ZFP131 maintain pluripotency by pausing POL II at pro-differentiation genes
Relations
BioSample SAMN16686428
SRA SRX9450252

Supplementary file Size Download File type/resource
GSM4886997_ES_day8_counts.txt.gz 226.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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